foods

Article Stress Condition on a Restricted Sodium Diet Using Substance (L-Glutamate) in a Pilot Randomized Cross-Over Study

Tamami Iwamoto 1,* , Andrea Wakita 2, Saiko Shikanai 3, Hideki Matsumoto 2, Mariko Hirota 2, Hisayuki Uneyama 2, Vu Thi Thu Hien 4 and Shigeru Yamamoto 1

1 Department of Food and Nutritional, Faculty of Human Life, Jumonji University, Saitama 352-8510, Japan; [email protected] 2 Co., Inc., Kanagawa 210-8681, Japan; [email protected] (A.W.); [email protected] (H.M.); [email protected] (M.H.); [email protected] (H.U.) 3 Asian Nutrition and Food Culture Research Center, Saitama 352-8510, Japan; [email protected] 4 National Institute of Nutrition, Hanoi 84, Vietnam; [email protected] * Correspondence: [email protected]; Tel.: +81-48-477-0555; Fax: +81-48-478-9367

Abstract: Hypertensive patients who adopt a sodium-restricted diet have difficulty maintaining this change, and this could increase stress. On the other hand, soup rich in umami substances () was reported to reduce indexes of anxiety and stress. The objective of this study was to measure mood and physiological stress indexes during administration of a sodium-restricted diet with and without   an umami substance (free L-glutamate) by a cross-over randomized, single-blind, placebo-controlled trial in Japanese female university students. The baseline was measured for 5 days followed by Citation: Iwamoto, T.; Wakita, A.; a sodium-restricted diet intervention phase that lasted for 10 days. The Profile of Mood States Shikanai, S.; Matsumoto, H.; Hirota, questionnaire was administered, a stress marker in saliva (chromogranin-A) was measured, and the M.; Uneyama, H.; Hien, V.T.T.; amount of sodium intake was confirmed from 24 h urine collection samples. Results showed that Yamamoto, S. Stress Condition on a the sodium reduction was verified by 24 h urine excretion. The percentage of change in the stress Restricted Sodium Diet Using Umami marker from the baseline showed that the stress level in group without the umami substance was Substance (L-Glutamate) in a Pilot significantly higher than that in the group with the umami substance (p = 0.013) after receiving a Randomized Cross-Over Study. Foods 2021, 10, 1739. https://doi.org/ sodium-reduced diet for 6 or more days, indicating that stress was alleviated. This study suggested 10.3390/foods10081739 that umami substances might help to ameliorate stress during a sodium-reduced diet, especially in the initial phase. Academic Editor: Jean-Xavier Guinard Keywords: stress; umami substances; restricted sodium diet; cross-over

Received: 21 June 2021 Accepted: 25 July 2021 Published: 28 July 2021 1. Introduction COVID-19, a new type of infectious disease, reminded us that prevention of non- Publisher’s Note: MDPI stays neutral communicable diseases (NCDs) is an issue that we should solve beyond the boundaries with regard to jurisdictional claims in of emerging and developing countries [1]. In 2013, WHO established the NCD Global published maps and institutional affil- Monitoring Framework and set seven goals for NCD prevention [2]. Among them, the only iations. specific nutrient intake target is salt reduction, and it calls on member countries to aim for a 30% reduction in salt intake from 2011 to 2025. Since then, many initiatives on salt reduction have been launched, accelerating the global salt-reduction movement. However, as of 2020, no country has achieved this goal [3]. For instance, the Japanese National Copyright: © 2021 by the authors. Nutrition and Health Survey 2019 reported that even though salt intake in Japanese was Licensee MDPI, Basel, Switzerland. reduced from 10.7 to 10.1 g/day during the period 2009–2019 [4], the intake is still almost This article is an open access article twice as much as the WHO recommendations of less than 5 g per day [5]. distributed under the terms and The latest analysis report of the Global Burden of Disease (GBD) has just been pub- conditions of the Creative Commons lished in The Lancet. As of 2019, primary care management and reductions in salt intake are Attribution (CC BY) license (https:// known to be potentially effective in reducing the burden of critical risk factors [6]. However, creativecommons.org/licenses/by/ 4.0/). salt not only improves the taste of food but is also used to preserve and flavor foods and to

Foods 2021, 10, 1739. https://doi.org/10.3390/foods10081739 https://www.mdpi.com/journal/foods Foods 2021, 10, 1739 2 of 11

improve yeast fermentation in dough [7–9], making a reduction a major challenge for the food industry. The committee on strategies to reduce sodium intake in the United State sug- gested the possibility of replacing some of the salt in food with other tastes or flavors [10]. An example is the addition of umami taste ingredients, dominantly composed of a building block of dietary protein, L-glutamate (an amino acid). Free L-glutamate can maintain food palatability with a lowered overall sodium amount in a food item [11–14]. In fact, free L-glutamate is being used in sodium-restricted diets such as for schizophreniform patients in hospital [15]. On the other hand, Kawano et al. have demonstrated that hypertensive patients who receive a sodium-restricted diet after diagnosis have difficulty maintaining the diet [16]; thus, in these subjects, having to continue with the diet could increase stress. Previous studies [17–20] showed improvement in mood state when subjects have an intake of dried bonito broth (dashi), which is rich in umami substances. For instance, in a randomized placebo-controlled human trial, it was found that dashi improved mood states and improved concentration significantly [17]. Another study reported that dashi not only improved the mood state but also decreased 8-hydroxy-20-deoxyguanosine, an oxidative stress marker [19]. This evidence may indicate the possibility of umami taste substances having a positive benefit for healthy eating. Several authors have reported the relationship between diet and stress; most of these studies are on the effects of psy- chological/physiological stress on dietary choices [21,22] but not on the effect of having several days of a sodium-reduced diet on stress in human studies. Our hypothesis is that an umami substance could be used as an approach to enhance the taste and palatability of a sodium-restricted diet and reduce psychological and physiological stress. Therefore, we investigated the effects of sodium-reduced-diet intake on the measurement of mood using the Profile of Mood States (POMS) questionnaire and measured physiological stress by testing the concentration of a saliva sympathetic system marker, chromogranin-A (CgA pmol/mg protein), during a sodium-restricted diet with and without the addition of umami substances in the foods. It is well known that salivary CgA (pmol/mg protein) changes more rapidly and more sensitively to psychological stressors than salivary cortisol does [23,24], and it has the advantage for the participants of being a noninvasive method.

2. Materials and Methods This study was approved by the Ethics Committees of Jumonji University (2012-008 and 2012-008-2), and Ajinomoto Co., Inc. (2012-008). Written consent was obtained from all subjects. This study was registered at UMIN-CTR Clinical Trial (UMIN000020514).

2.1. Subjects Thirty-one Japanese female university students were recruited by voluntary participa- tion. Women between 18 and 35 years old, with a regular menstrual cycle, who were not pregnant or lactating, without a habit of exhausting exercise, without chronic diseases, and without food allergies participated in the study.

2.2. Study Design It was a cross-over randomized, single-blind, placebo-controlled trial. Participants were randomly divided into group A or group B and assigned to start a sodium-restricted diet without an umami substance as the control group (group A) or a sodium-restricted diet with an umami substance as the Glu group (group B) for ten days. Before dividing into the two groups, participants received a diet with more than 4000 mg of sodium for 5 days as the baseline phase to standardize the sodium intake before the intervention phase. This amount of sodium was close to the Japanese diary intake [4].

2.3. Diet Intervention During the 5-day baseline and 10-day intervention phases, researchers provided participants with breakfast, lunch, dinner, snacks, and beverages. For snacks and beverages, the same brand of each item was purchased. Participants took the whole meals to eat Foods 2021, 10, 1739 3 of 11

at home or university, and they were instructed to eat all their food during the baseline and intervention phases, respectively. Snacks were optional. They were given a diary file to record all uneaten food portions (example: 1/4 main dish not eaten), snacks eaten, approximate amount of urine missed during collection, and comments regarding the study or whether their mood/physical condition was different from usual. The meals for lunch and dinner for the baseline and intervention were on 4-day menu cycles. The meals for breakfast and snacks were prepared or bought by Jumonji University. Main and side dishes for lunch and dinner were prepared for each participant in individual bags by the food factory Anshin Koubo Ltd., Yamagata, Japan, following standardized recipes and stored at −20 ◦C. The main and side dish portions were the same for all participants, but a dietitian adjusted individual dietary energy requirements by increasing or decreasing the amount of plain white rice and snacks. Energy and nutrients were calculated using the Excel Eiyokun software Ver. 6.0 (Kenpakusha Co., Ltd., Tokyo, Japan), based on the table of nutrient contents published by the Japanese Science and Technology Agency (5th edition). Before starting the study, randomized food samples from all diets (baseline, sodium-restricted diet without and with umami substance) were taken to analyze free L-glutamate, sodium, and potassium content at the Ajinomoto Co., Inc., laboratory. The free L-glutamate was measured by an amino acid analyzer (Hitachi L-8800 amino acid analyzer, Tokyo, Japan). Free L-glutamate intake by participants was calculated according to the data obtained at the laboratory of Ajinomoto Co., Inc. Sodium and potassium in the food were analyzed by inductively coupled plasma–atomic emission spectroscopy (ICPS-8100, Shimadzu). During the baseline, subjects received meals with sodium content higher than 4000 mg per day. In the intervention phase, subjects received a diet close to 2000 mg of sodium per day with a low amount of free L-glutamate intake for the control group, and high free L-glutamate for the umami group. The amounts of sodium and free L-glutamate used in the foods were controlled by the quantity of substances used, such as salt and umami seasonings, respectively. Subjects were not allowed to drink alcohol, coffee, or soft drinks with caffeine during the study. Before starting with the study, participants practiced all the procedures for taking samples (such as urine and saliva) and filling out the questionnaire (POMS).

2.4. Profile of Mood States (POMS) The full version of the POMS questionnaire consists of 65 items that are used to evaluate the mood state as follows: tension–anxiety, depression, anger–hostility, vigor, fatigue, and confusion. Subjects assessed moods they experienced the previous week on a five-point scale (0: not at all; 1: a little; 2: moderately; 3: quite a bit; 4: extremely frequent). The POMS—Japanese version published by Kaneko Shobo Co, Ltd. (Tokyo, Japan) was used as the test sheet. For the baseline, mood was assessed on the morning of day 1 (D1) and the morning of D1 of the intervention. These data were considered as the last data for the baseline phase. Since POMS reflects the mood state of the last week, for the intervention phase, it was conducted on the morning of D6 and the following morning after the completion of the intervention.

2.5. Anthropometric Data Height was self-reported using as reference the annual health check conducted at Jumonji University. Body weight, body muscle, and fat were measured by a bioelectrical impedance analysis system (InBody 2.0, Biospace Co., Ltd., Seoul, Korea). Subjects were asked to wear light clothing and to try to wear the same or similar clothes at each measure- ment. In addition, they removed shoes and socks to be measured by the InBody machine, they raised their arms 45◦ from the body and lightly pressed the metallic button until the measurement was completed. During the baseline phase, measurements were taken at D1 and the morning of D1 of the intervention. These data were considered as the last data for Foods 2021, 10, 1739 4 of 11

the baseline phase. For the intervention phase, anthropometric measurements were carried out on day 6 (D6) and the following morning after the completion of the intervention.

2.6. Saliva Sampling and Chromogranin-A Analyses Saliva samples were collected between 7 and 9 a.m. before breakfast. For baseline, it was collected on D1 and the morning of D1 of the intervention as the last data for the baseline phase. During the intervention phase, the samples were taken on day 3 (D3), D6, day 8 (D8), and the following morning after the completion of the intervention. Saliva samples were obtained using a saliva collector; the subjects chewed the cotton for three minutes, and then deposited it in a sample tube (Salivette, Sarstedt, Germany). The tubes were stored at −20 ◦C until centrifugation (3000 ppm, 5 min at 5 ◦C), and after centrifugation, they were stored again at −80 ◦C. The concentration of salivary chromogranin (Cg)-A (pmol/mg protein) was determined by Yanaihara Institute Inc. (Shizuoka, Japan).

2.7. Twenty-Four-Hour Urine Collection Urine samples were collected by subjects with special devices, the U-Container, a precise urine measurement device by Akita Sumitomo Bakelite Co., Ltd. (Akita, Japan), which is easy to handle. The first urine of the day was discarded, and collection was started from the second collection until the 24 h finish time. In case of a missing urine sample, participants had to record in their diary file the approximate amount of urine missed during the collection. The 24 h urine samples were taken on D1 and D5 of the baseline and D5 and D10 for the intervention phase. After finishing collection of the sample for the day, subjects brought it to Jumonji University, where it was stored at 5 ◦C, the total amount per day was calculated, and two samples of 10 mL were taken and stored at −20 ◦C until analysis. The amounts of sodium and potassium in the urine were analyzed at a laboratory testing service (Biomedical Laboratories, BML, Inc. (Saitama branch, Tokyo, Japan).

2.8. Statistical Analysis Data are presented as means and standard deviations (SD), and they were analyzed at baseline and, for the sodium-restricted diets, for D1–5 and D6–10 in the control group and the Glu group. Levene’s test was used to test homogeneity of variance. A paired-sample t-test was conducted to compare means of energy and nutrient intakes during the baseline and the sodium-restricted diet of the control group, and the baseline and sodium-restricted of the Glu group; and to compare anthropometric measurements between the control and the Glu groups for D1–5 and D6–10. The stress marker (CgA pmol/mg protein) was calculated as the percentage of change from baseline to the intervention phase D1–5 or D6–10 for both sodium-restricted groups. Differences between D1–5 and D6–10 within groups, and differences between the control and the Glu groups for D1–5 and D6–10 were conducted using a paired sample t-test. For urine and potassium excretion and POMS, a repeated-measure MANOVA was conducted to compare changes from baseline to D1–5 and for D6–10 followed by Tukey HSD post hoc test by controlling the random effect of the subjects when MANOVA showed differences. In data for which Levene’s test showed heterogeneity of variance, Wilcoxon t-test and Kruskal–Wallis (urine sodium excretions) were used. All statistical analyses were performed using JMP® 14.2.0 (SAS Institute Inc., Cary, NC, USA). A value of p < 0.05 was considered significant. For statistical analysis, “n” was the sum sample size.

3. Results Of the total 31 students, the stress marker and POMS analyses were performed on 23 participants (Figure1), because 3 dropped out after developing flu during the intervention phase and 5 subjects had incomplete data. Anthropometric data, of only 22 subjects were considered because of incomplete data. The adherence to the diet was assessed by urinary sodium excretion, daily record forms, and face-to-face interviews each Foods 2021, 10, 1739 5 of 11

time participants picked up meals to take home or brought the urine samples to Jumonji University.

Figure 1. CONSORT diagram showing the flow of participants through each stage of the study.

The dietary composition of sodium, potassium, and free L-glutamate is shown in Table1, according to the food analyses at the laboratory of Ajinomoto Co., Inc. The baseline diet had 4612 ±177 mg/d of sodium, and the sodium-restricted diet of the control group had 1993 ± 235 mg/d and the Glu group had 2048 ± 250 mg/d of sodium.

Table 1. Characteristics of the diet for the baseline and the sodium-restricted diets.

Sodium-Restricted Diets Baseline Diet Control Group Glu Group Mean SD Mean SD Mean SD Free L- g/day 0.61 0.02 0.36 0.06 1.10 0.65 Glutamate Sodium mg/day 4612 177 1993 235 2048 250 Potassium mg/day 2807 656 2149 209 1984 37 Values are mean and the standard deviation (SD) of the free-L-glutamate, sodium, and potassium in the food. Breakfast and snacks are included. Control group: sodium-restricted diet without umami substance. Glu group: sodium-restricted diet with umami substance (L-glutamate).

Table2 shows the energy and nutrient intake during the baseline and during both sodium-restricted diets calculated by Excel Eiyokun software. Free L-glutamate intake was calculated according to the data obtained at the laboratory of Ajinomoto Co., Inc. (Kanagawa, Japan) The energy intake was lower in the sodium-restricted diet of the control group (7310 ± 905 kJ/day, p = 0.006) and the Glu group (7332 ± 923 kJ/day, p < 0.026) when these were compared with the baseline diet (7502 ± 819 kJ/day). Energy intake decreased with a slight reduction of protein (the control group) and fat intake (the control and the Glu groups). Foods 2021, 10, 1739 6 of 11

Table 2. Energy and nutrient intakes during baseline and the sodium-restricted diets.

Sodium-Restricted Diet Baseline Diet Control Group Glu Group Mean SD Mean SD Mean SD Energy kJ/day 7502 819 7310 905 ** 7334 923 * Protein g/day 77 4 76 3 * 76 4 Fat g/day 47 2 45 1 ** 45 1 ** Carbohydrate g/day 267 44 262 50 263 50 Free g/day 0.9 0.1 0.3 0.0 ** 1.5 0.1 ** L-Glutamate Values are mean and standard deviation (SD) (n = 23). Statistical analyses were performed by paired-sample t-test between baseline and the control group, and baseline and the Glu group. * p < 0.05, ** p < 0.01.

The amount of urine excreted decreased from baseline to D1–5 and during D6–10 for both sodium-restricted diets (Table3). The sodium was reduced significantly from baseline to D1–5 (52%) and to D6–10 (44%). The potassium excreted in urine decreased slightly in the control group (Table3).

Table 3. Amount of urine, sodium, potassium, and chloride excreted in the baseline and in the sodium-reduced diets.

Sodium-Restricted Diet Sodium-Restricted Diet Baseline (Control Group) Baseline (Glu Group) D1–5 D6–10 D1–5 D6–10 Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Urine mL 1102 484 976 437 838 370 ** 1084 441 997 415 881 434 * Sodium mg/day 2282 879 1149 464 ** 981 426 ** 2176 704 1178 470 ** 958 383 ** Potassium mg/day 1369 435 1345 509 1168 441 * 1348 420 1272 441 1363 544 Chlorine mg/day 4013 1533 2073 868 ** 1817 772 ** 3839 1310 1948 892 ** 1639 641 ** Values are mean and standard deviations (SD) (n = 23). Statistical analyses were performed by repeated-measures MANOVA followed by Tukey HSD when statistically significant differences were found. * p < 0.05, ** p < 0.01.

Anthropology data showed a slight difference between the sodium-restricted diet in the control and the Glu groups in BMI on D1–5 (p = 0.027) and in muscle amount on D6–10 (p = 0.047) (Table4). Other anthropometric variables did not show a difference between the two sodium-restricted diet groups.

Table 4. Anthropometric measurements during the sodium-restricted diets.

D1–5 D6–10 Control Group Glu Groupp-Value Control Group Glu Group p-Value Mean SD Mean SD Mean SD Mean SD BMI kg/m2 20.7 2.3 20.5 2.2 0.027 20.6 2.2 20.4 2.2 0.053 Body water kg 25.1 3.3 25.0 3.0 0.286 25.3 3.2 25.1 3.1 0.039 Muscle kg 34.3 4.5 34.2 4.1 0.391 34.5 4.3 34.3 4.2 0.047 Body fat kg 14.7 3.7 14.2 3.7 0.095 14.2 3.5 13.9 3.6 0.246 Body fat % 28.5 4.9 27.8 4.9 0.163 27.9 4.5 27.4 4.8 0.304 Values are mean and standard deviations (SDs) (n = 22). Statistical analyses were performed by paired-sample t-test. Of 31 subjects, 3 dropped out, and 6 subjects’ data were deleted because of incomplete data.

The stress marker (CgA pmol/mg protein) calculated as the percentage of change from baseline to the intervention phase D1–5 or D6–10 was significantly higher in the sodium-restricted diet of the control group in phase D6–10 than in the sodium-restricted diet of the Glu group (p = 0.013) (Figure2). The difference between D1–5 and D6–10 was that the stress marker tended to increase in the control group, but it was not significant statically (p = 0.052). Foods 2021, 10, 1739 7 of 11

POMS did not show any difference during either sodium-restricted diet (Supplemen- tary Table S1).

Figure 2. Stress condition marker (CgA pmol/mg protein) represented as percentage (%) of change from baseline to D1–5 or D6–10 in the sodium-restricted diet control group (white) and Glu group (black) and its standard deviation. In D6–10 CgA (pmol/mg protein) value as stress condition showed that Glu group value was significantly lower than control group diet by paired sample t-test (n = 23) (p = 0.013).

4. Discussion This (pilot) study suggests an important finding for healthy eating. In the late stage of the sodium-reduced diet (D6–D10), the change in saliva concentration of chromogranin-A was significantly higher than the baseline in the control group (Figure2). On the other hand, the amount of sodium excretion in urine dropped during the intervention phase with the sodium-restricted diet (Table3). This sodium reduction in the body by the continuous intake of a sodium-restricted diet could induce psychological stress. However, this psychological stress appears only as a physiological response, in the CgA (pmol/mg protein) and not in the POMS questionnaire, so its conscious effect may be weak. To our knowledge, this is the first study to show that having a sodium-reduced diet for several days can induce a physiological stress response as shown by our control group. Kawano et al. indicates that hypertensive patients have a higher salt craving when receiving a sodium-reduced diet than healthy individuals without sodium restrictions [16], so this may be worth confirming through a similar study in hypertensive patients. In our study, when an umami substance was added in the food, psychological stress was lower than in the control group with a sodium-reduced diet. Since the latest brain science studies using NIRS have shown that umami substances enhance salty-like acceptance [25], many scientists have shown that umami substances enhance the taste of sodium-reduced foods in different food groups [11,26–29]. Based on this evidence, the increase in psychological stress due to sodium-reduced-diet intake is probably related to the fact that it is difficult to switch to a sodium-reduced diet from a normal diet (due to factors such as a decrease in palatability). It seems that the acceptability of a low-salt diet can be increased with umami fortification to reduce psychological stress. Decreased palatability due to a decrease in the “deliciousness” of a low-salt diet not only makes it difficult for healthy people to adopt a diet with lower sodium intake to prevent health risks such as cardiovascular diseases, but it is also an issue in improving the quality of life (QOL) of hospitalized patients, especially in malnourished elderly patients. In a recent study, sodium reduction in hospital meals showed a lower eating rate for hospitalized elderly patients, leading to poor nutritional status [30]. It has been shown Foods 2021, 10, 1739 8 of 11

that even in hospitalized patients, the eating rate of inpatients’ meals (sodium-reduced diet) in the psychiatric elderly is improved by the addition of umami seasonings to daily meals [15]. Positive usage of umami substances in a sodium-reduced diet is likely to be more helpful in improving the nutrition of inpatients. In our study, the energy intake was decreased by a slight reduction of protein (control group) and fat intake (control and Glu groups) during intervention, but these reductions may not have been enough to produce a physiological effect, since the reductions were 192 and 162 kJ/d in the sodium-restricted control and Glu groups compared to baseline, respectively. We suggest that in the sodium-restricted diet the need to balance the tastes of salty foods with plain white rice could be reduced, and as consequence, the subjects could not finish the whole serving of rice and some side dishes. Jeffery et al. found that participants preferred to decrease the intake of sodium by increasing naturally lowsodium foods, such as fruits and vegetables, than by eating low-sodium processed foods, which were considered less palatable [31]. In our study, the processed foods were eaten for breakfast. These foods kept their original sodium content. The sodium intake from the processed food, and therefore its sodium, was controlled by instructing subjects about the amount of food to consume before starting the study. The amounts of free glutamate in the sodium-restricted control and Glu groups were controlled by the addition of sources of free glutamate, such as umami seasoning. The 24 h urinary sodium excretion showed a reduction from the baseline to the inter- vention phase, which could suggest some physiological changes in the body in adapting to this reduction of sodium, such as increases in plasma renin activity and aldosterone [32]. The adherence to the diet was assessed by urinary sodium, daily record forms, and face- to-face interviews each time participants picked up meals to take home or brought the urine samples to Jumonji University. We could demonstrate that, in our population, a sodium-restricted diet caused stress as measured by the stress markers, but mood did not change as measured by POMS, though it has been reported by Ishizaki and Kuroda that dashi, which is rich in umami substances, could improve the mood state as measured by POMS [17–19]. As mentioned above, CgA (pmol/mg protein) increased in the sodium-restricted diet control group but did not change in the sodium-restricted diet Glu group. This could suggest that participants of the sodium-restricted diet Glu group could adapt better to the diet, as Mattes reported as a reason for accepting a lower-sodium diet [33], but these adaptations could occur physiologically without changing their mood. Our hypothesis concerning the increase in CgA (pmol/mg protein) in the sodium-restricted control group is that even though most of the participants did not realize which meals were the ones with umami seasoning, the information is not detected consciously but maybe it was perceived by the glutamate receptors in the mouth and in the gastro-intestinal tract [34,35] and transmitted to the brain; hence, it may not change the amount of the stress marker during the sodium-restricted diet of the Glu group. It has been suggested that after taste cognition in the cortex of the brain, taste information is further processed in the limbic system in terms of emotion. The outcome is finally conveyed to the hypothalamus for the regulation of feeding [36]. In an animal study, it was reported that the oral administration of 2% of dashi for 29 days reduced anxiety, which could be the result of changing amino acid levels in the brain [37]. In our study, the variability of CgA (pmol/mg protein) (Figure2) was slightly large; however, the paired-sample t-test showed a strong significant difference between the control and the Glu group (p = 0.013). A previous study in young healthy subjects showed a large standard deviation [38]. CgA may vary among subjects; however, it has two advantages: it is a noninvasive test, and each participant can take the saliva sample at home. To our knowledge, this is the first study carried out in humans to study the effect of umami seasoning on stress during a reduced-sodium diet. However, the study has some limitations. First, there was no questionnaire item regarding diet acceptability or sensory difference of the sodium-restricted control and Glu groups. However, during interviews, Foods 2021, 10, 1739 9 of 11

participants commented that they did not perceive the differences between the two sodium- reduced diets. Second, it is possible that students could have had an academic examination during the intervention phase, which could influence stress and mood state. However, this was a cross-over study, thus this bias should be minimized. Third, subjects were Japanese female students. Food preferences can be significantly influenced by food culture, and it may be necessary to consider whether the results of female students raised in the Japanese food culture rich in umami sources are applicable to Western food culture. However, this limitation may not be significant because the finding that the sodium-reduced foods’ palatability is improved by fortifying with umami taste is reported by several studies in different subjects from different food cultures [39–42].

5. Conclusions The present study suggested that an umami substance, free L-glutamate, could ame- liorate stress during a reduced-sodium diet. To achieve the maximum amount of sodium suggested by WHO without changing the stress indexes, free L-glutamate could be a tool to encourage consumers to make healthier choices without sacrificing palatability. These results may contribute to the development of strategies to achieve the sodium-reduction goals by using seasonings rich in umami taste.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/foods10081739/s1, Table S1: POMS score during baseline and sodium restricted diet. Author Contributions: T.I. designed the study, collected and analyzed the data, interpreted the statistical analyses, and wrote the manuscript with the assistance of S.S. and V.T.T.H. S.Y. supervised the study. A.W. and H.U. contributed to the literature review. H.M. and M.H. developed the protocol for the sodium, potassium, and free L-glutamate analyses of food samples. T.I. had primary responsibility for the final content of the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Ajinomoto Co., Inc. Institutional Review Board Statement: This study was approved by the Ethics Committees of Jumonji University (2012-008 and 2012-008-2), and Ajinomoto Co., Inc. (2012-008). Informed Consent Statement: Written consent was obtained from all subjects. Data Availability Statement: This study was registered at UMIN-CTR Clinical Trial (UMIN000020514). Acknowledgments: The authors gratefully acknowledge the cooperation of all participants. They thank Andrew Durkin of Indiana University for helping with the manuscript. Conflicts of Interest: T.I., S.S., H.V.T.T., and S.Y. had no conflict of interest. A.W., H.M., M.H., and H.U. are employees of Ajinomoto Co., Inc. The funders had no role in the design of the study and statistical analyses or interpretation of data in the writing of the manuscript or in the decision to publish the results. Funders proposed the protocol for the sodium, potassium, and free L-glutamate analyses of food samples.

References 1. Belanger, M.J.; Hill, M.A.; Angelidi, A.M.; Dalamaga, M.; Sowers, J.R.; Christos SMantzoros, C.S. COVID-19 and disparities in nutrition and obesity. N. Engl. J. Med. 2020, 383, e69. [CrossRef][PubMed] 2. World Health Organization. A Comprehensive Global Monitoring Framework Including Indicators and a Set of Voluntary Global Targets for the Prevention and Control of Non-Communicable Diseases; WHO: Geneva, Switzerland, 2012. 3. Global Nutrition Report 2020. Available online: https://globalnutritionreport.org/reports/2020-global-nutrition-report/ (ac- cessed on 25 May 2021). 4. Ministry of Health, Labor and Welfare. Annual Report of the National Health and Nutrition Survey in 2019. Tokyo (In Japanese). Available online: https://www.mhlw.go.jp/content/000710991.pdf (accessed on 25 May 2021). 5. World Health Organization. Prevention of Cardiovascular Disease: Guidelines for Assessment and Management of Cardiovascular Risk; World Health Organization: Geneva, Switzerland, 2007. 6. GBD 2019 Risk Factors Collaborators. Global burden of 87 risk factors in 204 countries and territories, 1990–2019: A systematic analysis for the Global Burden of Disease Study 2019. Lancet 2020, 396, 1223–1249. [CrossRef] Foods 2021, 10, 1739 10 of 11

7. Taormina, P.J. Implications of salt and sodium reduction on microbial food safety. Crit. Rev. Food Sci. Nutr. 2010, 50, 209–227. [CrossRef] 8. Belcastro, G.; Rastelli, E.; Mariotti, V.; Consiglio, C.; Facchini, F.; Bonfigliolo, B. Continuity or discontinuity of the life-style in central Italy during the Roman Imperial Age-Early Middle Ages transition: Diet, health, and behavior. Am. J. Phys. Anthropol. 2007, 132, 381–394. [CrossRef] 9. Belz, M.C.; Ryan, L.A.; Arendt, E.K. The impact of salt reduction in bread: A review. Crit. Rev. Food Sci. Nutr. 2012, 52, 514–524. [CrossRef][PubMed] 10. Institute of Medicine (US) Committee on Strategies to Reduce Sodium Intake. Taste and Roles of Sodium in Foods: A Unique Challenge to Reducing Sodium Intake. In Strategies to Reduce Sodium Intake in the United States; Henney, J.E., Taylor, C.L., Boon, C.S., Eds.; National Academies Press (US): Washington, DC, USA, 2012. 11. Yamaguchi, S.; Takahashi, C. Interactions of and sodium chloride on saltiness and palatability of a clear soup. J. Food Sci. 1984, 49, 82–85. [CrossRef] 12. Atung, T.; Demirag, K. Influence of monosodium glutamate on flavor acceptability and on the reduction of sodium chloride in some ready-made soup. Chem. Mikrobiol. Technol. Lebensm. 1993, 15, 161–164. 13. Ishida, M.; Tezuka, H.; Hasegawa, T.; Cao, L.; Imada, T.; Kimura, E.; Matsumoto, H.; Kawano, R.; Arai, H. Sensory evaluation of a low-salt menu created with umami, similar to savory substance. J. Jpn. Soc. Food Nutr. 2011, 64, 305–311. [CrossRef] 14. Hayabuchi, H.; Morita, R.; Ohta, M.; Nanri, A.; Matsumoto, H.; Fujitani, S.; Yoshida, S.; Ito, S.; Sakima, A.; Takase, H.; et al. Validation of preferred salt concentration in soup based on a randomized blinded experiment in multiple regions in Japan—influence of umami (L-glutamate) on saltiness and palatability of low-salt solutions. Hypertens Res. 2020, 43, 525–533. [CrossRef] 15. Kawano, R.; Ishida, M.; Kimura, E.; Matsumoto, H.; Arai, H. Pilot intervention study of a low-salt diet with monomagnesium di-L-glutamate as an umami seasoning in psychiatric inpatients. Psychogeriatrics 2015, 15, 38–42. [CrossRef] 16. Kawano, Y.; Ando, K.; Matsuura, H.; Tsuchihashi, T.; Fujita, T.; Ueshima, H. Report of the working group for dietary salt reduction of the Japanese society of hypertension: (1) rationale for salt restriction and salt-restriction target level for the management of hypertension. Hypertens. Res. 2007, 10, 879–886. [CrossRef][PubMed] 17. Ishizaki, T.; Hisano, M.; Umeki, Y.; Kuroda, M.; Hayabuchi, H. The evaluation of skipjack soup stock consumption on mood states using questionnaire (POMS). J. Study Dietary Habit. 2005, 16, 39–43. [CrossRef] 18. Ishizaki, T.; Kuroda, M.; Sugita, M. The effect of dried skipjack soup stock on mood and emotional states, especially the fatigue state. J. Jpn. Soc. Food Sci. Tech. 2006, 53, 225–228. [CrossRef] 19. Nozawa, Y.; Ishizaki, T.; Kuroda, M.; Noguchi, T. Effect of dried-bonito broth intake on peripheral blood flow, mood, and oxidative stress marker in humans. Physiol. Behav. 2008, 93, 267. [CrossRef][PubMed] 20. Kuroda, M.; Ishizaki, T.; Maruyama, T.; Takatsuka, Y.; Kuboki, T. Effect of dried-bonito broth on mental fatigue and mental task performance in subjects with a high fatigue score. Physiol. Behav. 2007, 92, 957–962. [CrossRef][PubMed] 21. Khaled, K.; Tsofliou, F.; Hundley, V.; Helmreich, R.; Almilaji, O. Perceived stress and diet quality in women of reproductive age: A systematic review and meta-analysis. Nutr. J. 2020, 19, 92. [CrossRef][PubMed] 22. Leng, G.; Adan, R.A.H.; Belot, M.; Brunstrom, J.M.; de Graaf, K.; Dickson, S.L.; Hare, T.; Maier, S.; Menzies, J.; Preissl, H.; et al. The determinants of food choice. Proc. Nutr. Soc. 2017, 76, 316–327. [CrossRef] 23. Nakane, H.; Asami, O.; Yamada, Y.; Ohira, H. Salivary chromogranin A as index of psychosomatic response. Biomed. Res. 1998, 19, 401–406. [CrossRef] 24. Den, R.; Toda, T.; Nagasawa, S.; Kitamura, K.; Morimoto, K. Circadian rhythm of human salivary chromogranin A. Biomed. Res. 2007, 28, 57–60. [CrossRef][PubMed] 25. Onuma, T.; Maruyama, H.; Sakai, N. Enhancement of saltiness perception by monosodium glutamate taste and soy sauce odor: A Near-Infrared Spectroscopy study. Chem. Senses 2018, 43, 151–167. [CrossRef][PubMed] 26. Jinap, S.; Hajeb, P.; Karim, R.; Norliana, S.; Yibadatihan, S.; Abdul-Kadir, R. Reduction of sodium content in spicy soups using monosodium glutamate. Food Nutr. Res. 2016, 60, 30463. [CrossRef] 27. Ball, P.; Woodward, D.; Beard, T.; Shoobridge, A.; Ferrier, M. Calcium diglutamate improves taste characteristics of lower-salt soup. Eur. J. Clin. Nutr. 2002, 56, 519–523. [CrossRef] 28. Rodrigues, J.F.; Gonçalves, C.S.; Pereira, R.C.; Carneiro, J.D.S.; Pinheiro, A.C.M. Utilization of temporal dominance of sensations and time intensity methodology for development of low-sodium Mozzarella cheese using a mixture of salts. J. Dairy Sci. 2014, 97, 4733–4744. [CrossRef][PubMed] 29. Dos Santos, B.A.; Campagnol, P.C.B.; Morgano, M.A.; Pollonio, M.A.R. Monosodium glutamate, disodium inosinate, , lysine and taurine improve the sensory quality of fermented cooked sausages with 50% and 75% replacement of NaCl with KCl. Meat. Sci. 2014, 96, 509–513. [CrossRef][PubMed] 30. Hirose, K.; Tran, T.P.; Yamamoto, S. Decreasing salt in hospital meals reduced energy intake in elderly Japanese inpatients. J. Nutr. Sci. Vitaminol. 2021, 67, 105–110. [CrossRef] 31. Jeffery, R.W.; Pirie, P.L.; Elmer, P.J.; Bjornson-Benson, W.M.; Mullenbach, V.A.; Kurth, C.L.; Johnson, S.L. Low sodium-high potassium diet: Feasibility and palatability in a normotensive population. Am. J. Pub. Health 1984, 74, 492–494. [CrossRef] 32. He, F.J.; Markandu, N.D.; Sagnella, G.A.; MacGregor, G.A. Effect of salt intake on renal excretion of water in humans. Hypertension 2001, 38, 317–320. [CrossRef][PubMed] Foods 2021, 10, 1739 11 of 11

33. Mattes, R.D. The taste for salt in humans. Am. J. Clin. Nutr. 1997, 65, 692S–697S. [CrossRef] 34. Chaudhari, N.; Landin, A.M.; Roper, S.D. A metabotropic glutamate receptor variant functions as a taste receptor. Nat. Neurosci. 2000, 3, 113–119. [CrossRef] 35. Uneyama, H. Nutritional and physiological significance of luminal glutamate-sensing in the gastrointestinal functions. Yakugaku Zasshi 2011, 131, 1699–1709. [CrossRef] 36. Yamamoto, T. Central mechanisms of taste: Cognition, emotion and taste-elicited behaviors. Jpn. Dent. Sc. Rev. 2008, 44, 91–99. [CrossRef] 37. Funatsu, S.; Kondoh, T.; Kawase, T.; Ikeda, H.; Nagasawa, M.; Michael Denbow, D.; Furuse, M. Long-term consumption of dried bonito dashi (a traditional Japanese fish stock) reduces anxiety and modifies central amino acid levels in rats. Nutr. Neurosci. 2015, 18, 256–264. [CrossRef] 38. Toda, M.; Ichikawa, H. Effect of laughter on salivary flow rates and levels of chromogranin A in young adults and elderly people. Environ. Health Prev. Med. 2012, 17, 494–499. [CrossRef][PubMed] 39. Hoppu, U.; Hopia, A.; Pohjanheimo, T.; Rotola-Pukkila, M.; Mäkinen, S.; Pihlanto, A.; Sandell, M. Effect of salt reduction on consumer acceptance and sensory quality of food. Foods 2017, 6, 103. [CrossRef][PubMed] 40. Morita, R.; Ohta, M.; Umeki, Y.; Nanri, A.; Tsuchihashi, T.; Hayabuchi, H. Effect of monosodium glutamate on saltiness and palatability ratings of low-salt solutions in Japanese adults according to their early salt exposure or salty taste preference. Nutrients 2021, 13, 577. [CrossRef][PubMed] 41. Rocha, R.A.R.; Ribeiro, M.N.; Silva, G.A.; Rocha, L.C.R.; Pinheiro, A.C.M.; Nunes, C.A.; Carneiro, J.D.S. Temporal profile of flavor enhancers MAG, MSG, GMP, and IMP, and their ability to enhance salty taste, in different reductions of sodium chloride. J. Food Sci. 2020, 85, 1565–1575. [CrossRef] 42. Leong, J.; Kasamatsu, C.; Ong, E.; Hoi, J.T.; Loong, M.N. A study on sensory properties of sodium reduction and replacement in Asian food using difference-from–control test. Food Sci. Nutr. 2015, 4, 469–478. [CrossRef]